E16ラット胚の子宮内での脳室内注入とエレクトロポレーションで

Hi, my name is William Ti.I'm from the Arnold Creek Stein Lab. Today, myself and others are gonna be showing you intrauterine injection of E 16 rat embryos. Hi, my name is Laura Elias.

Today I'm gonna be showing you how to prepare the microinjection pipettes and load the pipettes for the int ventricular injections and electroporation that we will be doing today. I'll also be showing you how to perform the interventricular injections and electroporation in E 16 rat embryos. So in utero, eject injections and electroporation in the rat are particularly useful because the red is a system where you cannot easily do genetic Manipulations.

Okay, so there's a number Of surgical tools that we use for this procedure. We use a freer, a couple of addin forceps, one, one pair that's serrated, and another pair that has a little tooth on the end. We use a pair of hemostats.

This is a pair of scissors, and then we use these two staplers. This is the larger ones that, that we use for rats. This is the smaller one that we use for mice, uses a size seven staple, and this is used as a size nine staple and they're just spring loaded.

Some of the things that we use here to assist with the surgeries, we just use a simple fiber optic light source with a double head on it. We use a heated pad that just is c circulates, warm, heated water through there to keep it warm. The working surface, we use this elective electroporation system made by BTX.

We have a couple different sizes of paddles that we use based on how big, how large the embryos are. The blue side is the positive Side. All right, so now we're going to bevel our injection pipettes.

And this is very important so that your, your pipette is, has a large enough opening so that you can load your DNA, but also that it's very, very sharp so that it doesn't damage the embryo when you inject. And if you damage the embryo when you inject, you're, you're gonna have a much higher rate of mortality. And so that's, that's not good.

So this is actually a very important process as in this surgery. And first we're gonna just put some water onto this beveling stone that's rotating here. And then we're going to take, let's see here, just a, a, a stick.

And we're going to place our pipette that we just pulled onto this stick with some tape. And we're gonna use one of these micro manipulators to hold the pipette on the bevel and allow it to bevel here. So we'll just place this and we use microscope in order to visualize the tip of the pipette.

And we'll just place it right down in the water on top of the bevel earlier so that there's a little bit of pressure. And you can see the tip pressing against the, the, the stone here. And allow it to bevel for some time for at least, you know, 15 minutes.

I like to leave them even for, you know, an hour. Sometimes they're just, they get really, really sharp, but it definitely depends person by person how long they bevel their pipettes. All right, so here we have, you can see on the top we have a pipette that's been pulled that hasn't been beveled yet.

So while it's sharp, it doesn't have any opening through which you could load your, your DNA. Now the bottom pipette has been beveled, and you can see this nice angled opening Now that will allow you to load your DNA, but it still has a sharp tip so that you can inject without creating any damage. All right, so now we're gonna fill our injection pipettes with DNA.

So it's important that you prepare your DNA in an endotoxin free manner so that you don't inject any of the lipopolysaccharides when you inject your DNA. So we're gonna take our DNA and actually here, get our paraform ready. So you're gonna just make your paraform here and you're gonna wanna collect your DNA and just place it as a little dot on this param.

And then we're gonna take, so I usually take for every 10 microliters of my DNA, which I usually use at about 1.5 to 1.7 micrograms per a microliter. I'll use about one microliter of this fast cream dye. And this is just to help us localize our injection and make sure that we've successfully injected into the ventricle.

So then I'll just mix this fast green dye with my DNA. And then you take your injection electrode and we'll just be filling the pipette by pulling back on the plunger. And you can see here if it, if the pipette correctly and has a big of enough opening, the DNI should load pretty easily.

Sometimes you need to just move the plunger back and forth to help it load up correctly. And so then you can load the pipette like this. And so we have the DNA loaded fully into this pipette and it's ready for Injection.

Okay, so I'm Gonna anesthetize the animal. We use these dec capi cones. I just kind of roll the lip of it back to make, make it easier to hold.

So let's begin. I usually just kind of put it on the edge of the cage to make a nice little tunnel for him to go into like that. Okay, this animal looks like it's down.

We're gonna go ahead and do a toe pinch to confirm that and just need to grab the toe, give it a good pinch. Not too hard. You don't want to injure the animal, but just to make sure it's down.

And this animal is definitely down. So I'm gonna go ahead and shave her belly. I just wanna avoid the tet so that you don't snip off the ends of them.

I think it could be very painful for the animal. So the animal is shaved. Now I'm gonna take it into the hood.

We're gonna wash her up and begin the procedure. Okay, so we've confirmed that this animal is out once again and we're ready to wash her up first. What I want to do is put a little eye ointment on, and that's just to prevent her eyes from drying up each eye.

And then what we're gonna do is alternating wash with alcohol prep pads and iodine pads. We do that three times, three alternating wipes. We have to be very gentle so that you don't damage the embryos while you're doing this.

So you want to just kind of swipe straight down, just wait straight back. This is number one. I just gotta get her nice and wet first so that I can do the wipes.Good.

That's two and that's three. Okay, so we've put this cover on it to help with steril and now I'm gonna go ahead and open up the animal. So let's cut.

Alright, first thing that I need to do is put on the ster. We put on sterile Gloves and we're ready to open up here. Always want to confirm that the animal's still down.

So I'll do another toe pinch and we'll be doing those throughout the procedure. Usually what I do is I go ahead and pinch a little bit of the tissue. I make a quick incision, grab it at the top and slice down, grab the other pair of the forceps, hold the tissue apart.

Then you grab the muscle you want, cut right on the midline so that you don't hear any vessels or nerves. Be careful not to grab Sure. So it looks like we have a nice yield here.

I wanna make sure and keep it well lubricated so that it doesn't desiccate. And I'll do another token just to be sure we're good and looks like we're good. So I always position these For Laura, and what I usually do is have, or what I always do is have the head on the position it so that the embryo's head is on the mother rat's, right side hallways just for uniformity.

And so I'll start now. So here we can see the embryo. We can see the, the tail and the hind lens here, and the head further up here.

So here we're looking at the head of this embryo. You can see the midline here at this dark line. You can see the left hemisphere and the right hemisphere, as well as there's a hind brain.

Now I'm gonna be targeting the left hemisphere in the injection. Pipette inject, pull up a bit and inject the dye. So now you see it's spreading into the left lateral ventricle.

And so that looks like a successful injection. All right, so now we see that the ventricle have filled up nicely. The dye is actually passed from the left to the right ventricle.

So we have D and DNA in both ventricles. At this point, I'm going to take my electroporation electrodes and place them across the brain with a positive electrode on the left side. So here, so that the DNA will enter the progenitors and the cortical wall of the left hemisphere.

And now I'm passing the current through. And when we start passing the electro current, you see some foam on the negative end of the electrode. And this is reassuring and tells you that you've made the Correct contacts.

Okay, now that we've electro these embryos, I'm gonna go ahead and put them back in and sew the out. I'm going to now give her 10 mils of antibiotic surgical suture. I like to use block stitches.

After that, we're gonna staple the skin with the skin stapler.Okay? Okay.So I've given her a dose of buprenorphine to make sure that she doesn't experience any pain when she comes out. And now I'll just put her in the incubator and that'll keep her warm while she's coming out, just to make sure she doesn't experience any hypothermia.

Okay, So we've just shown you how to perform in utero, intraventricular injections, and electroporation. In the rat, you can inject a number of different things. You can simply inject a virus, including lentivirus or retrovirus directly into the ventricular.

And in this case, you won't need to do the electroporation because the virus will integrate into the, into the cells. If you inject plasmid, DNA plasmids, as we did today, you will have to perform the electroporation on in order to get the plasmids into the cells lining the ventricle. So you, you can use these vectors for a number of of possibilities.

You can use them just to simply follow the cells and express GFP, and then you're able to watch these progenitors divide and migrate. You can also use these vectors to manipulate the cells. So you can express S-H-R-N-A hairpins, and thus you can knock down specific genes.

You can also over express specific genes, and in this way, you can manipulate the cells that you've electroporated or infected with the virus. And this can be a very powerful technique to study the role of specific proteins in the process that you're interested in. And this is specifically a very important for studies in the rat because you're not able to make genetic manipulations as a rat as easily as you are able to in the mouse.